Pluripotent human stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), offer the prospect to regenerate myocardium after an infarct. To date, however, cell transplantation studies have yielded only small grafts of human cardiac muscle. Recent studies in our group and elsewhere indicate that the inclusion of stromal and vascular cells, along with cardiomyocytes, is much more effective in generating new myocardium. By transplanting pre-vascularized, engineered human myocardium, we have generated myocardial grafts that are as thick as the infarcted wall. The current proposal builds on these observations, using human cardiovascular progenitors and differentiated cells in hydrogel-based tissue engineering, to more fully regenerate the heart. In Aim 1 we will test the ability of cardiovascular mesodermal progenitors to give rise to new myocardium in vitro and to repair the infarcted rat heart in vivo. The progenitors will be compared to tri-cell constructs containing hESC-derived cardiomyocytes, endothelial cells and MSCs. We hypothesize that the mesodermal progenitors will have enhanced proliferative capacity and the ability to differentiate according to environmental cues, resulting in larger grafts with greater vascular density and better integration with the host. Aim 2 tests the responses of engineered human heart tissues to conditioning regimens of cyclic strain, electrical pacing, and varying the stiffness of their hydrogel matrix. The effects on tissue structure and electromechanical function will be assessed in vitro, and optimally conditioned constructs will be compared with unconditioned constructs for their ability to repair the infarcted heart. We also will use diffusion tensor MRI to determine if pre-aligning cardiac fibers in tissue constructs influences final fiber orientation after transplantation in the heart. These experiments will be the first to study responses of ex vivo human myocardium to changes in mechanical load or rate, and thus should provide a useful system for basic science and therapeutic screens. In Aim 3, our optimized tissue engineering approach will be compared head- to-head vs. cell therapy, to determine which offers more promise for cardiac regeneration and restoration of ventricular function. In addition to providing important pre-clinical therapeutic information, these experiments will offer insights into fundamental aspects of human cardiovascular development.